JPH0529231B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Field of Application] The present invention relates to a method for purifying phosphatidylinositol.

[Conventional technology]

Phospholipids are widely distributed in animals and plants, and serve various physiological functions. For this reason, phospholipids are used as therapeutic agents for arteriosclerosis, hyperlipidemia, liver disease, heart disease, etc., and as emulsifiers for foods, cosmetics, pharmaceuticals, and the like.

Normally, phospholipids are obtained by separating and purifying oils and fats from plant seeds as a gum, but the composition of the phospholipids contained differs depending on the type of seed material. Normally, the phospholipid composition in plant seeds is phosphatidylcholine (hereinafter referred to as PC).
Phosphatidylethanolamine (hereinafter referred to as PE), phosphatidylserine (hereinafter referred to as PS), and phosphatidylinositol (hereinafter referred to as
The main component is PI), which is extremely difficult to separate using current technology, so they are often used as a mixture.

Conventional methods for fractionating phospholipids include concentrating the target substance using acetone or alcohol, and then performing chromatography using silicic acid, alumina, etc. In particular, when isolating PI, phospholipids are treated with isopropanol to increase the concentration of PI, and then fractionated by chromatography using a silicate column (for example, Biochemistry Experimental Structure 3, Lipid Chemistry p.278).

[Problem that the invention seeks to solve]

However, in such conventional methods, it is necessary to perform column chromatography two or more times using a silicic acid column and an alumina column, and the operation is complicated and requires a lot of labor and time.
In addition to low efficiency, there were problems in that highly pure PI could not be obtained and PI could not be purified on an industrial scale.

The purpose of the present invention is to propose a method for purifying PI that can efficiently purify PI to a high degree of purity.

[Means for solving problems]

The present invention involves adsorbing phosphatidylinositol onto a polysaccharide-based anion exchanger, and removing phosphatidyl from the anion exchanger using an organic solvent containing a buffer salt that has a higher affinity for the anion exchanger than phosphatidylinositol. This is a method for purifying phosphatidylinositol, which is characterized by eluting inositol.

The phospholipids to be purified in the present invention may be derived from animals, plants, or microorganisms. The phospholipid to be purified may be a phospholipid fraction extracted with an organic solvent such as chloroform or methanol, but it is also possible to extract and remove other phospholipids such as PC and PE with an alcohol such as methanol, ethanol, or propanol. A crude PI fraction obtained by concentrating PI is preferable.

The polysaccharide-based anion exchanger used for purification in the present invention is a carrier made of a natural crosslinked polysaccharide polymer such as cellulose, agarose, or dextran, to which anion exchange groups are attached as dissociative groups, and the amount of exchange groups is per 1 g. Preferably 0.1 to 0.5 meq.
Examples of such anion exchangers include aminoethyl groups, diethylaminoethyl groups, triethylaminoethyl groups, guanidoethyl groups, para-aminobenzyl groups, etc. introduced into substituents of polymer chains such as cellulose, agarose, and crosslinked dextran. Cross-linked cellulose, agarose, dextran, etc. with triethanolamine conjugated with glyceryl or polyglyceryl groups; Benzoylated diethylaminoethyl cellulose or diethylaminoethyl agarose; Benzoylated-naphthoylated diethylaminoethyl cellulose or diethylaminoethyl agarose ; cellulose or agarose;
Those commonly used in ion exchange chromatography can be used, such as weakly phosphorylated cellulose or agarose with polyethyleneimine adsorbed.

Such an anion exchanger is swollen in advance with an organic solvent, activated by contacting with a buffer salt, and then brought into contact with the substance to be purified in the presence of an organic solvent to adsorb PI. The method for contacting the anion exchanger with the product to be purified may be a batch method in which the product to be purified and the anion exchanger are mixed and stirred in the presence of an organic solvent, but it is also possible to use a batch method in which the anion exchanger is packed in a column and dissolved in the solvent. A continuous method may be used in which the product to be purified is passed through the column and brought into contact with the solution.

The buffer salt used for activation of the anion exchanger can be the same as that used for elution in the subsequent step, such as alkali salts such as acetic acid, formic acid, propionic acid, ammonium salts, amine salts, etc. be. The activation method involves bringing an aqueous solution of these buffer salts into contact with an anion exchanger, separating it from the liquid, removing water with methanol, etc., and then swelling the anion exchanger with an organic solvent.

The same organic solvent can be used for swelling and for dissolving the product to be purified, such as chloroform, methanol, ethanol, isopropanol, butanol, acetone, acetonitrile, dichloromethane, carbon tetrachloride, etc. alone or in combination. Although it can be used, mixed solvents containing polar solvents are preferred.

By bringing the product to be purified into contact with an anion exchanger in this manner, PI and other phospholipids are adsorbed onto the anion exchanger. Therefore, by separating the solvent from the anion exchanger and washing the anion exchanger with fresh solvent, other phospholipids adsorbed on the anion exchanger can be completely removed.

By contacting the anion exchanger thus separated with an organic solvent containing a buffer salt, the adsorbed PI can be eluted and recovered with high purity. Batsuhua salt has a higher affinity (adsorption) for anion exchangers than PI, and the above-mentioned salt used for activation can be used, and a small amount of water may be added to improve solubility. . Furthermore, the same organic solvents as mentioned above can be used.

The PI purification method will be specifically explained below using the batch method as an example. First, an anion exchanger is activated by a conventional method, swollen with a chloroform-methanol solvent [10:0 to 0:10 (v/v)] solution, and the swollen anion exchanger and the solvent are combined.
Mix at a ratio of 10:1 to 1:20 (v/v).
On the other hand, the crude PI fraction obtained from the raw material phospholipids was added to chloroform, dichloroethane, dichloromethane,
Dissolved in a halogenated hydrocarbon such as carbon tetrachloride and added to the mixture of the anion exchanger and solvent,
Stir between 0 and 40°C. The stirring time can be determined as appropriate, but is preferably about 15 minutes to 2 hours. thus
The anion exchanger adsorbed with PI was filtered and diluted with chloroform-methanol solvent (10:0 to 0:10).
(v/v)) and a buffer salt such as sodium acetate or ammonium acetate at a concentration of 0.1 to 0.5% (w/v) is used to elute and purify only PI.

In the case of a column-passing method, an anion exchanger is packed in a column and the anion exchanger is passed through the column under conditions similar to those described above to perform adsorption and elution. Although the PI thus obtained is highly pure, it may be subjected to further purification.

〔Effect of the invention〕

According to the present invention, PI is purified by adsorbing it to a polysaccharide-based anion exchanger, so that PI can be purified in large quantities, with high purity, and in high yields with simple operations and with little labor and time. Purification can be carried out.

〔Example〕

Examples of the present invention will be described below.

Example 1 14 g of lipid extracted from 1 kg of dry yeast with chloroform-methanol (1:1 (v/v)) was washed with 1 part of isopropanol to obtain 3 g of crude PI fraction.

On the other hand, DEAE-Sepharose, a polysaccharide-based anion exchanger with diethylaminoethyl groups introduced into cellulose, CL-6B (manufactured by Pharmacia, trademark) 100
ml was suspended in 1N sodium acetate (PH7.0), filtered, water was added, stirred gently and filtered, methanol was added, stirred gently and filtered, and chloroform-methanol (1:4 (v /v))
Pretreated.

2 g of the above crude PI fraction was added to 100 ml of the pretreated solvent-containing anion exchanger, and the mixture was stirred at room temperature for 1 hour to be adsorbed. This was filtered and washed with 100 ml of chloroform-methanol (1:4 (v/v)) to obtain fraction 1 (F ₁ ). Next, it was washed with a 0.1% sodium acetate (w/v) solution 1 of chloroform-methanol (1:4 (v/v)) to obtain Flakfuyon 2 (F ₂ ).

Thin layer chromatography (TLC) of concentrated and desalted fractions 1 and 2.
As a result of analysis, fraction 1 contained 1 g of lipids, including PS and other phospholipids other than PI, and fraction 2 contained 800 mg of PI alone.

The TLC diagram of each fraction is shown in Figure 1.

Example 2 20 g of pasty soybean phospholipid was washed with 1 part of isopropanol to obtain 3 g of crude PI fraction.

On the other hand, a polysaccharide-based anion exchanger TEAE-cellulose (manufactured by Selva Fine Biochemica, trademark) in which triethylaminoethyl groups were introduced into cellulose was pretreated in the same manner as in Example 1, and the crude PI fraction was obtained. Adsorption was carried out in the same manner as in Example 1.

Then chloroform-methanol (1:1.1)
(v/v)) Wash with 100ml, fraction 1 (F ₁ )
I got it. Next, chloroform-methanol (1:1
(v/v)) of 0.3% ammonium acetate (w/v)
Washing with solution 1 yielded fraction 2 (F ₂ ).

Fraction 1 contained 1 g of lipids other than PI, and fraction 2 contained 1.8 g of PI alone.

The TLC diagram of each fraction is shown in Figure 2.

Example 3 50 ml of a polysaccharide-based anion exchanger QAE-Sephadex (manufactured by Pharmacia, trademark) in which a diethyl-(2-hydroxypropyl) aminoethyl group was introduced into cross-linked dextran was pretreated in the same manner as in Example 1. 1 g of Folch fraction I (manufactured by Sigma, containing PI 10-20%) of extracted lipids from bovine brain was adsorbed in the same manner as in Example 1, and then filtered and mixed with chloroform-methanol (1:1 (v/v)). After washing with 100 ml, fraction 1 (F ₁ ) was obtained. Then 0.1% of chloroform-methanol (1:4 (v/v))
Wash with 500 ml of ammonium acetate (w/v) solution;
Fraction 2 (F ₂ ) was obtained.

As a result of analyzing the concentrated and desalted fractions 1 and 2, it was found that fraction 1 contained
Fraction 2 contained 500 mg of phospholipids other than PI, and 120 mg of PI alone.

The TLC diagram of each fraction is shown in Figure 3.

Comparative Example Silicic acid was suspended in chloroform and packed into a column, and phospholipids were fractionated by chromatography. First, a crude product obtained in the same manner as in Example 1 was prepared.
3 g of the PI fraction was spread on a silicic acid column (φ4.5 x 32 cm), and then eluted with a mixed solvent of chloroform-methanol, with the methanol concentration being 15%, 25%,
Elution was performed by increasing the concentration sequentially to 30%, 40%, 50%, and 80%. The amount of solvent to be flowed was 2, 4, 2,
1, 2, and 2 were collected in 20 to 200 ml portions to obtain 50 fractions. Each fraction was combined and concentrated and analyzed by TLC. the result,
Even if chromatography is performed by increasing the polarity of the elution solvent, sufficient separation cannot be achieved, and the PI
It was also very difficult to separate PS and PS.

The TLC diagram of each fraction is shown in Figure 4.

[Brief explanation of the drawing]

FIGS. 1 to 4 are TLC diagrams showing the results of Examples 1 to 3 and Comparative Example, respectively.

[Claims]

linear equation The bis(diphenylphosphino)amine represented by [representing an iodine atom] is reacted with a Grignard reagent represented by the following formula RX [] [wherein R represents an alkyl group or a benzyl group, and X represents a chlorine, bromine or iodine atom]. characterized by reacting a halide compound represented by the following formula: [In the formula, R and X have the same meanings as above]